This invention relates to modulating the growth of multicellular aggregates (e.g., tumors).
In tissue engineering, the successful development of a cell-based implant for restoring or improving body function through the production and/or secretion of bioactive substances is dependent upon the growth state and spatial organization of cells within the implant. For example, hepatocytes that are cultured in vitro in a three-dimensional configuration retain a more differentiated state and display more liver-specific functions than do cells grown in a two dimensional system. Similarly, the control of growth rates is of importance in biochemical engineering processes and bioreactor applications.
The invention derives from the discovery that the macroscopic growth of multicellular aggregates, such as tumors, is modulated by solid stress (i.e., pressure exerted by solids, rather than fluids). Although solid stress inhibits the growth of such aggregates on a macroscopic level, continued cell proliferation and a decrease in apoptosis results in compaction of cells within the multicellular aggregate.
The invention thus features a method for controlling the growth of a multicellular aggregate in vitro. The method entails: (i) embedding a plurality of cells in a growth matrix, (ii) measuring the level of solid stress on the cells, (iii) modulating the level of solid stress on cells within the growth matrix, and allowing the cells to grow within the growth matrix, thereby forming a multicellular aggregate, and thereby controlling the growth of the multicellular aggregate in vitro.
The invention also provides a method for producing a multicellular aggregate having a pre-selected size; the method entails: embedding a plurality of cells in a growth matrix, and allowing the cells to grow within the matrix, wherein the growth matrix exerts a degree of solid stress on the cells adequate to achieve a multicellular aggregate of the pre-selected size. In a variation of the method, one can produce a multicellular aggregate having a pre-selected size and shape. Typically, this method is carried out by embedding a plurality of cells in a growth matrix, and allowing the cells to grow within the matrix, wherein the growth matrix is contained within a vessel (e.g., a vessel that is non-uniform in shape), and the growth matrix together with the vessel exert a degree of solid stress (e.g., non-isotropic stress) on the cells adequate to achieve a multicellular aggregate of the pre-selected size and shape. These methods can be used to produce artificial tissues such as livers, skin, muscle, bone, and various other organs.
The cells may be tumor cells (e.g., from muscle, liver, colon, or mammary tumors) or non-tumor cells, such as healthy, wild-type cells. The cells can be derived from an established cell line, or they can be primary cells. Examples of preferred cell types include, without limitation, liver cells, pancreatic cells, brain cells, skin cells, muscle cells, mammary cells, and bone cells.
A variety of growth matrices having differing mechanical strengths may be used in the invention. For example, the cells can be grown in a matrix containing agarose, for example at a concentration of 0.3% to 2.0% (w/v). Alternatively, the cells may be grown in a matrix containing collagen, with or without a glycosaminoglycan such as hyaluronic acid. In another variation of this method, the growth matrix may contain alginate. In addition to containing a compound for producing a growth matrix having stiffness (e.g., agarose), the growth matrix contains nutrients for growing the cells. For convenience, conventional cell culture media can be used to dissolve the matrix-forming compound (e.g., agarose) and provide nutrients to the cells.
As shown by the examples provided below, multicellular aggregates that are grown in a non-isotropic stress field preferentially grow in the direction of the least stress. Thus, the invention also provides a method for modulating the growth pattern of a multicellular aggregate. This method entails embedding a plurality of cells in a growth matrix in which solid stress exerted by the matrix is non-isotropic and thereby defines a template which modulates the growth pattern of the multicellular aggregate. By allowing the cells to grow within the matrix, a multicellular aggregate is formed, having a shape that is dictated by the non-isotropic stress field. Such a method therefore can be used to produce multicellular aggregates of virtually any desired shape (e.g., physiologically relevant shapes, such as those of organs, or portions thereof, or shapes convenient for grafting or implantation).
In a variation of the methods described above, the invention provides a method for identifying a therapeutic compound for treating a multicellular aggregate. As described below, compounds that decrease solid stress exerted by multicellular aggregates are expected to provide a beneficial therapeutic effect by inhibiting collapse of vascular and lymphatic vessels within the multicellular aggregate, thereby facilitating blood flow and delivery of therapeutics throughout aggregates, and facilitating lymphatic drainage of tumors. This method for identifying therapeutic compounds entails:
embedding a plurality of cells in a growth matrix,
allowing the cells to grow within the growth matrix, thereby forming a multicellular aggregate,
treating the multicellular aggregate with a test compound, and
measuring a decrease in the level of solid stress on the multicellular aggregate following treatment with the test compound, relative to the level of solid stress prior to treatment, as an indication that the test compound is a therapeutic compound for treating the multicellular aggregate (i.e., as a reliever of solid stress).
In related aspect, the invention provides a method for treating a multicellular aggregate in a mammal. In this method, a mammal (e.g., a human or a rodent, such as a mouse, in an animal model of a human disorder) is identified as being afflicted with a multicellular aggregate (e.g., a tumor), and solid stress exerted by the aggregate is relieved. Relief of solid stress can be accomplished, for example, by administering to the mammal an antibody that specifically binds an integrin, or an enzyme that dissolves the extracellular matrix (e.g., a collagenase, hyaluronidase, or protease). Alternatively, the extracellular matrix can be dissolved (and solid stress relieved) by topical treatment of the extracellular matrix with heat, ultrasound, microwaves, radiation, or the like.
By xe2x80x9csolid stressxe2x80x9d is meant pressure exerted on a solid by another solid, for example stress exerted on a multicellular aggregate by a growth matrix. Solid stress, therefore, is distinct from interstitial fluid pressure.
By xe2x80x9cmulticellular aggregatexe2x80x9d is meant a plurality of connected cells (e.g., as in a mass of tissue). The cells of such an aggregate may be tumorigenic or non-tumorigenic. They need not be, but can be, homogenous. Included are primary cells, as well as cells of established cell lines. If desired, the cells may be wild-type, mutated (naturally or intentionally), or genetically engineered to produce a recombinant gene product (e.g., a secreted protein).
The invention offers several advantages. The level of solid stress imposed on cells can readily be modulated by embedding and growing the cells in a growth matrix of a defined stiffness (i.e., gel strength). By controlling stress exerted on the multicellular aggregate, one can control the proliferation and apoptotic rates of cells (e.g., tumor cells) within the aggregate. By applying stress in a non-isotropic manner on the growing multicellular aggregate, one can shape the multicellular aggregate into nearly any shape. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.